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Kirenol relieves rheumatoid arthritis by targeting the TWEAK/Fn14 pathway

  • Authors:
    • Zixin Chen
    • Jinxuan Wang
    • Lijuan Xiao
    • Zhihui Chen
    • Wenchuan Luo
    • Wen Xu
    • Ya Lin
    • Mei Huang
    • Yuqian Zhang
    • Yinghao Wang
    • Yaping Chen
    • Lihong Nan
  • View Affiliations / Copyright

    Affiliations: College of Pharmacy, Fujian University of Traditional Chinese Medicine, Fuzhou, Fujian 350122, P.R. China, School of Basic Medical Sciences, Fujian Medical University, Fuzhou, Fujian 350004, P.R. China
    Copyright: © Chen et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 145
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    Published online on: July 14, 2025
       https://doi.org/10.3892/ijmm.2025.5586
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Abstract

Fibroblast‑like synoviocytes (FLSs) are the primary drivers of synovial tissue hyperplasia in rheumatoid arthritis (RA). Activation of the tumor necrosis factor‑like weak inducer of apoptosis (TWEAK)/fibroblast growth factor‑inducible immediate‑early response protein 14 (Fn14) pathway significantly contributes to the pathogenesis of RA. Kirenol (Kir), a compound with anti‑inflammatory and antirheumatic properties, has an unclear mechanism of action. To comprehensively investigate the effects and potential mechanisms of Kir on RA, the present study employed both an in vitro model of transforming growth factor‑β1 (TGF‑β1)‑induced human fibroblast‑like MH7A synoviocytes proliferation and an in vivo collagen‑induced arthritis (CIA) rat model. The effects of Kir on synovial fibroblasts were detected via flow cytometry, ELISA, hematoxylin and eosin staining, safranin‑O/fast green staining, immunohistochemistry, immunofluorescence and western blotting. Kir ameliorated pathological damage in the synovial tissue of CIA rats, suppressed rheumatoid factor production, regulated the T helper 17 cells/regulatory T cell balance and mitigated joint inflammation and swelling. Additionally, Kir markedly downregulated the protein levels of the TWEAK/Fn14 pathway in synovial tissue. Surface plasmon resonance demonstrated that Kir could specifically bind to Fn14. Kir significantly suppressed the TGF‑β1‑mediated aberrant proliferation and migration of MH7A cells. However, the overexpression of Fn14 reversed the inhibitory effects of Kir on the abnormal proliferation and migration of cells, as did the activation of the TWEAK/Fn14 pathway. These results suggest that Kir possesses anti‑RA properties by inhibiting abnormal immune‑inflammatory responses, as well as synovial cell proliferation and migration. These effects of Kir may be linked to a decrease in the activity of the TWEAK/Fn14 pathway.
View Figures

Figure 1

Schedule of the animal
experiment.

Figure 2

Ameliorative effects of Kir on
rheumatoid arthritis in CIA rats. (A) Foot swelling of the rats in
each group after Kir intervention. (B) Paw edema of the rats in
each group. (C) AI of the rats in each group. (D) RF levels in the
serum of the rats in each group. (E) Histopathological changes in
the synovial tissue in the rat joints (H&E staining;
magnification, ×200); the white arrows indicate vascular dilation
and congestion and the yellow arrows indicate inflammatory cell
infiltration. (F) Histopathological changes of the cartilage tissue
in the rat joints (safranin-O/fast staining; magnification, ×50);
cartilage tissue is stained in red and bone tissue in green; black
arrows indicate the invasion of synovial tissue and articular
cartilage destruction. All data are expressed as the mean ± SD
(n=10). Kruskal-Wallis followed by Dunn's post-test was performed
in C, one-way ANOVA followed by Tukey test was performed in D and
one-way ANOVA followed by Games-Howell method in B.
**P<0.01 vs. the control group;
#P<0.05, ##P<0.01 vs. the CIA group.
Kir, Kirenol; CIA, collagen-induced arthritis; AI, arthritis index;
RF, rheumatoid factor; H&E, hematoxylin and eosin; Pre,
prednisone.

Figure 3

Kir inhibits inflammatory factors and
MMP-13 in the synovial tissues of CIA rats. Representative
immunohistochemical images of (A) MCP-1, (B) ICAM-1 and (C) MMP-13
in the synovial tissues of various rats (magnification, ×200).
Quantitative analysis of the immunohistochemistry of (D) MCP-1, (E)
ICAM-1 and (F) MMP-13 in the synovial tissues of various groups of
rats. Representative images and quantification of the western blot
results of (G) CXCL-10 and (H) CXCR3 protein expression. All data
are expressed as the mean ± SD (n=5). Kruskal-Wallis with Dunn's
post-test was performed in D, one-way ANOVA with Games-Howell
method in E and F and one-way ANOVA followed by Tukey test was
performed in G and H. *P<0.05, **P<0.01
vs. the control group; #P<0.05,
##P<0.01 vs. the CIA group. Kir, Kirenol; CIA,
collagen-induced arthritis; MCP-1, monocyte chemoattractant
protein-1; ICAM-1, intercellular cell adhesion molecule-1; MMP-13,
matrix metalloproteinases 13; CXCL-10, C-X-C motif chemokine
ligand-10; CXCR3, C-X-C motif chemokine receptor 3.

Figure 4

Kir mediates the differentiation of
CD4+ T cells in CIA rats. (A) Representative flow
cytometry charts of CD4+IL-17+ cells in
peripheral blood. (B) Representative flow cytometry charts of
CD4+CD25+Foxp3+ Treg cells in
peripheral blood. (C) Flow cytometric analysis of the proportion of
Th17+ cells. (D) Flow cytometric analysis of the
proportion of Treg cells. (E) The ratios of Th17/Treg cells in
peripheral blood were analyzed. (F) IL-10 and (G) IL-17 levels in
serum were determined via ELISA. All the data are expressed as the
mean ± SD (n=5). One-way ANOVA followed by Tukey test was performed
in all analyses. **P<0.01 vs. the control group;
#P<0.05, ##P<0.01 vs. the CIA group.
Kir, Kirenol; CIA, collagen-induced arthritis; Th17, T helper cell
17; IL-17, interleukin 17; IL-10, interleukin 10.

Figure 5

Kir attenuates the proliferation and
migration of TGF-β1-induced MH7A cells. (A) Viability of MH7A cells
treated with different concentrations of Kir. (B) Effects of Kir on
the TGF-β1-induced proliferation of MH7A cells. (C and D) Effects
of Kir on the TGF-β1-mediated migration of MH7A cells
(magnification, ×50). All data are expressed as the mean ± SD
(n=5). One-way ANOVA followed by Tukey test was performed in all
analyses. **P<0.01 vs. the control group;
#P<0.05, ##P<0.01 vs. the TGF-β1 group.
Kir, Kirenol; TGF-β1, transforming growth factor-β1.

Figure 6

Surface plasmon resonance measurement
of the binding between Kir and Fn14. (A) Sensorgram of Kir binding
to the Fn14-immobilized chip. (B) The fitted curve for different
concentrations of Kir bound to immobilized Fn14. Kir, Kirenol;
Fn14, fibroblast growth factor-inducible immediate-early response
protein 14; RU, response unit.

Figure 7

Fn14 overexpression abolishes the
effect of Kir on the proliferation and migration of TGF-β1-induced
MH7A cells. (A) The GFP expression in MH7A cells transfected with
the lentivirus (magnification, ×200). (B) The Fn14 protein levels
in MH7A cells transfected with Fn14-OE compared with NC lentivirus;
β-actin was used as normalization control; (n=3)
*P<0.05 vs. NC group. (D) Effect of Kir on the
proliferation of Fn14-overexpressing MH7A cells. (C and E) Effect
of Kir on the migratory ability of Fn14-overexpressing MH7A cells
(magnification, ×50); (n=5). One-way ANOVA followed by LSD was
performed in B, one-way ANOVA followed by Tukey test was performed
in D and one-way ANOVA followed by Games-Howell was performed in F.
**P<0.01 vs. the control group, #P<0.05
or ##P<0.01 vs. the TGF-β1 group,
△P<0.05 vs. the 5 µM Kir group. All the data
are expressed as the mean ± SD. Kir, Kirenol; TGF-β1, transforming
growth factor-β1. Kir, Kirenol; Fn14, fibroblast growth
factor-inducible immediate-early response protein 14; Fn14-OE,
Fn14-overexpressing lentivirus; NC, negative control; Con,
control.

Figure 8

Fn14 overexpression blocks the
effects of Kir on chemokines and MMP-13 in TGF-β1-induced MH7A
cells. Levels of (A) MCP-1, (B) ICAM-1, (C) MMP-13 and (D) CXCL-10
in the cell supernatant of MH7A cells. All the data are expressed
as the mean ± SD (n=5). One-way ANOVA followed by Tukey test was
performed in C and one-way ANOVA followed by Games-Howell in A, B
and D. **P<0.01 vs. the control group,
#P<0.05 or ##P<0.01 vs. the TGF-β1
group, △P<0.05 or △△P<0.01 vs. the 5
µM Kir group. Fn14, fibroblast growth factor-inducible
immediate-early response protein 14; Kir, kirenol; MCP-1, monocyte
chemoattractant protein-1; ICAM-1, intercellular cell adhesion
molecule-1; MMP-13, matrix metalloproteinases 13; CXCL10, C-X-C
motif chemokine ligand 10.

Figure 9

Kir attenuates the levels of
TWEAK/Fn14 pathway-associated proteins in the synovial tissues of
CIA rats. Representative western blots and quantification of the
(A) TWEAK, (B) Fn14, (C) TRAF2, (D) ASK1 and (E) NIK protein levels
and the phosphorylation of (F) IRE1, (G) IKKα and (H) p100. β-actin
was used as the loading control. All the data are expressed as the
mean ± SD (n=5). Kruskal-Wallis followed by Dunn's post-test was
performed in D, one-way ANOVA followed by Games-Howell in C and
one-way ANOVA followed by Tukey test was performed in A, B and E-H.
**P<0.01 vs. the control group, #P<0.05
or ##P<0.01 vs. the CIA group. Fn14, fibroblast
growth factor-inducible immediate-early response protein 14; Kir,
kirenol; CIA, collagen-induced arthritis; TWEAK, tumor necrosis
factor-like weak inducer of apoptosis; NIK, nuclear
factor-κB-inducing kinase; IRE1, inositol-requiring enzyme 1; IKKα,
inhibitor of κB kinase α.

Figure 10

Kir reduces nuclear localization of
p52 and RelB in the synovial tissues of CIA rats. (A)
Representative images of immunofluorescence staining for p52
protein in the nucleus (magnification, ×200). (B) Quantitative
analysis of nuclear p52 protein. (C) Representative images of
immunofluorescence staining for RelB proteins in the nucleus
(magnification, ×200); (D) Quantitative analysis of nuclear RelB
protein. All the data are expressed as the mean ± SD (n=5). One-way
ANOVA with Games-Howell method was performed for all analyses.
**P<0.01 vs. the control group, #P<0.05
or ##P<0.01 vs. the CIA group. Kir, kirenol; CIA,
collagen-induced arthritis; PCNA, proliferating cell nuclear
antigen.

Figure 11

Fn14 overexpression blocks the effect
of Kir on TWEAK/Fn14 pathway-related proteins in TGF-β1-induced
MH7A cells. Representative images and quantification of western
blot results showing the protein expression of (A) TWEAK (B) and
Fn14, and the phosphorylation of (C) IKKα and (D) p100. β-actin was
used as the loading control. Representative images and
quantification of western blot results for the nuclear proteins (E)
p52 and (F) RelB. PCNA was used as the loading control for
nucleoprotein. All the data are expressed as the mean ± SD (n=3).
One-way ANOVA followed by Tukey test was performed in all analyses.
*P<0.05 or **P<0.01 vs. the control
group, #P<0.05 vs. the TGF-β1 group,
△P<0.05 vs. the 5 µM Kir group. Kir, kirenol;
TGF-β1, transforming growth factor-β1; PCNA, proliferating cell
nuclear antigen; TWEAK, tumor necrosis factor-like weak inducer of
apoptosis; Fn14, fibroblast growth factor-inducible immediate-early
response protein 14; PCNA, proliferating cell nuclear antigen.

Figure 12

Kir relieves rheumatoid
arthritis-induced synovial cell injury via modulation of the
TWEAK/Fn14 signaling pathway in vivo and in vitro.
TWEAK, tumor necrosis factor-like weak inducer of apoptosis; Fn14,
fibroblast growth factor-inducible immediate-early response protein
14; RA, rheumatoid arthritis; NIK, nuclear factor-κB-inducing
kinase; IKKα, inhibitor of κB kinase α; MCP-1, monocyte
chemoattractant protein-1; ICAM-1, intercellular cell adhesion
molecule-1; MMP-13, matrix metalloproteinases 13; CXCL10, C-X-C
motif chemokine ligand 10; C-X-C motif chemokine receptor 3; IL-17,
interleukin 17; IL-10, interleukin 10.
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Copy and paste a formatted citation
Spandidos Publications style
Chen Z, Wang J, Xiao L, Chen Z, Luo W, Xu W, Lin Y, Huang M, Zhang Y, Wang Y, Wang Y, et al: Kirenol relieves rheumatoid arthritis by targeting the TWEAK/Fn14 pathway. Int J Mol Med 56: 145, 2025.
APA
Chen, Z., Wang, J., Xiao, L., Chen, Z., Luo, W., Xu, W. ... Nan, L. (2025). Kirenol relieves rheumatoid arthritis by targeting the TWEAK/Fn14 pathway. International Journal of Molecular Medicine, 56, 145. https://doi.org/10.3892/ijmm.2025.5586
MLA
Chen, Z., Wang, J., Xiao, L., Chen, Z., Luo, W., Xu, W., Lin, Y., Huang, M., Zhang, Y., Wang, Y., Chen, Y., Nan, L."Kirenol relieves rheumatoid arthritis by targeting the TWEAK/Fn14 pathway". International Journal of Molecular Medicine 56.4 (2025): 145.
Chicago
Chen, Z., Wang, J., Xiao, L., Chen, Z., Luo, W., Xu, W., Lin, Y., Huang, M., Zhang, Y., Wang, Y., Chen, Y., Nan, L."Kirenol relieves rheumatoid arthritis by targeting the TWEAK/Fn14 pathway". International Journal of Molecular Medicine 56, no. 4 (2025): 145. https://doi.org/10.3892/ijmm.2025.5586
Copy and paste a formatted citation
x
Spandidos Publications style
Chen Z, Wang J, Xiao L, Chen Z, Luo W, Xu W, Lin Y, Huang M, Zhang Y, Wang Y, Wang Y, et al: Kirenol relieves rheumatoid arthritis by targeting the TWEAK/Fn14 pathway. Int J Mol Med 56: 145, 2025.
APA
Chen, Z., Wang, J., Xiao, L., Chen, Z., Luo, W., Xu, W. ... Nan, L. (2025). Kirenol relieves rheumatoid arthritis by targeting the TWEAK/Fn14 pathway. International Journal of Molecular Medicine, 56, 145. https://doi.org/10.3892/ijmm.2025.5586
MLA
Chen, Z., Wang, J., Xiao, L., Chen, Z., Luo, W., Xu, W., Lin, Y., Huang, M., Zhang, Y., Wang, Y., Chen, Y., Nan, L."Kirenol relieves rheumatoid arthritis by targeting the TWEAK/Fn14 pathway". International Journal of Molecular Medicine 56.4 (2025): 145.
Chicago
Chen, Z., Wang, J., Xiao, L., Chen, Z., Luo, W., Xu, W., Lin, Y., Huang, M., Zhang, Y., Wang, Y., Chen, Y., Nan, L."Kirenol relieves rheumatoid arthritis by targeting the TWEAK/Fn14 pathway". International Journal of Molecular Medicine 56, no. 4 (2025): 145. https://doi.org/10.3892/ijmm.2025.5586
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